This Application is the National Stage of International Application Serial No. PCT/JP99/04766, filed Sep. 2, 1999.
The present invention relates to a novel ADAM protein.
An extracellular matrix is a cell-supporting tissue surrounding cells of the tissue and is composed of a fibrous protein such as collagen and elastin, a complex carbohydrate such as proteoglycan, a glycoprotein such as fibronectin and laminin, which relate to cell-adhesion, and a sugar such as hyaluronic acid. The exracellular matrix is known to have the important influence of activities of cells such as shape, metabolism, migration, proliferation and differentiation. Therefore, the extracellular matrix is known to be associated with many living body phenomena such as development, aging, inflammation, wound healing, immunity and tumor of the living body. It is known that the abnormal degradation of the extracellular matrix occurs in a variety of diseases such as rheumatoid arthritis, osteoarthritis, osteoporosis, cancer metastasis and infiltration, arteriosclerosis and corneae ulcer. There is a possibility that regulation of the enzyme activities involved in degradation of the extracellular matrix produces a therapeutic agent for these diseases.
ADAM (A disintegrin and metalloprotease) family proteins have the similar structure to that of hemorrhagic venom and are composed of a metalloprotease region and a disintegrin region. Many of ADAM family proteins are membrane proteins and 10 or more cDNAs have been isolated from a variety of organisms (T. G. Wolfsberg et al., Journal of Cell Biology 131:275-278, 1995). The physiological functions of ADAM proteins are known to be involved in cell fusion, cell differentiation, host defense and the like. That is, Fertilin, which is an ADAM expressed on a sperm, is associated with adhesion of an egg and sperm by binding to integrin xcex16xcex21 on an egg via a disintegrin region (D. Myles et al., Proc. Natl. Acad. Sci. USA 91:4195-4198, 1994). A disintegrin region of meltrin is reported to be associated with fusion of myocyte (T. Yagami-Hiromasaet al., Nature 377:652-656, 1995). In addition, KUZ, which is an ADAM protein of Drosophila is associated with differentiation of nerve (J. Rooke et al., Science 273:1227-1230, 1996). In addition, TNF-xcex1 convertase (R. A. Black et al, Nature 385:729-733, 1997) and ADAM 10 (C. A. Lunn et al., FEBS letter 40:333-335, 1997) are reported to cleave a TNF-xcex1 precursor to convert it into a secreted type.
New human-derived ADAM family proteins enable development of new medicines which have the activity of regulating the activities of those proteins and are useful for preventing or treating joint diseases such as a variety of diseases based on those activities, for example, rheumatoid arthritis and osteoarthritis. Therefore, there has been desired finding of human-derived novel ADAM proteins and development of a process for mass-production of them in the art of the present invention.
In order to solve the above problems, the present inventors studied intensively and, as a result, found an ADAM gene having a novel base sequence and found that an ADAM protein encoded by it has the protease activity and is involved in degradation of an extracellular matrix. Based on these findings, the present inventors further studied and, as a result, completed the present invention.
That is, the present invention provides:
(1) a protein or a salt thereof which has an amino acid identical to or substantially identical to an amino acid sequence represented by SEQ ID No: 2,
(2) the protein or a salt thereof according to the above (1), which has an amino acid identical to or substantially identical to an amino acid represented by SEQ ID No: 2 as a disintegrin region,
(3) the protein or a salt thereof according to the above (1), which belongs to an ADAM family,
(4) the protein or a salt thereof according to the above (1), which has an amino acid sequence identical or substantially identical to an amino acid sequence represented by SEQ ID No: 1 or SEQ ID No: 15,
(5) the protein or a salt thereof according to the above (1), which has the protease activity,
(6) a partial peptide of the protein according to the above (1), which has an amino acid sequence identical or substantially identical to an amino acid sequence represented by SEQ ID No: 6, or a salt thereof,
(7) a DNA which has a DNA having a base sequence encoding a protein having an amino acid sequence identical or substantially identical to an amino acid sequence represented by SEQ ID No: 2,
(8) the DNA according to the above (7), which has a base sequence represented by SEQ ID No: 3 or SEQ ID No: 16,
(9) a DNA which has a DNA encoding the partial peptide according to the above (6),
(10) the DNA according to the above (9), which has a base sequence represented by SEQ ID No: 4,
(11) a recombinant vector which has the DNA according to the above (7),
(12) a transformant transformed with the recombinant vector according to the above (11),
(13) a method for producing the protein or a salt according to the above (1), which comprises culturing the transformant according to the above (12) to produce the protein according to the above (1),
(14) an antibody against the protein or a salt thereof according to the above (1) or the partial peptide or a salt thereof according to the above (6),
(15) a diagnostic agent which comprises the DNA according to the above (7) or the antibody according to the above (14),
(16) an agent which comprises the protein or a salt thereof according to the above (1) or the partial peptide or a salt thereof according to the above (6),
(17) a medicine which comprises the protein or a salt thereof according to the above (1) or the partial peptide or a salt thereof according to the above (6),
(18) the medicine according to the above (17), which is an agent for preventing or treating disc herniation, ischialgia, glomerular nephritis, diabetic nephropathy, hepatic fibrosis, pulmonary fibrosis or osteopetrosis,
(19) a method for screening for a compound or a salt thereof which promotes or inhibits the protease activity, which comprises using the protein according to the above (1) or a salt thereof,
(20) a kit for screening for a compound or a salt thereof which promotes or inhibits the protease activity, which comprises the protein according to the above (1) or a salt thereof,
(21) a compound or a salt thereof which promotes or inhibits the protease activity and is obtainable by using the method for screening according to the above (19) or the kit for screening according to the above (20),
(22) a medicine which comprises a compound or a salt which promotes or inhibits the protease activity and is obtainable by the method for screening according to the above (19) or the kit for screening according to the above (20),
(23) an agent for degrading an extracellular matrix, which comprises a protein having an amino acid identical or substantially identical to an amino acid sequence represented by SEQ ID No: 5, or a salt thereof,
(24) the agent according to the above (23), wherein the extracellular matrix is a proteoglycan,
(25) the agent according to the above (23), which is a pharmaceutical composition,
(26) the agent according to the above (23), which is an agent for preventing or treating disc herniation, ischialgia, glomerular nephritis, diabetic nephropathy, hepatic fibrosis, pulmonary fibrosis or osteopetrosis,
(27) a method for screening for a compound or a salt thereof which promotes or inhibits the extracellular matrix degrading enzyme activity, which comprises using a protein having an amino acid sequence identical or substantially identical to an amino acid sequence represented by SEQ ID No: 5, or a salt thereof,
(28) a kit for screening for a compound or a salt thereof which promotes or inhibits the extracellular matrix degrading enzyme activity, which comprises a protein having an amino acid sequence identical or substantially identical to an amino acid sequence represented by SEQ ID No: 5, or a salt thereof,
(29) a compound or a salt thereof which promotes or inhibits the extracellular matrix degrading enzyme activity, which is obtainable by the method for screening according to the above (27) or the kit for screening according to the above (28),
(30) a medicine which comprises a compound or a salt thereof which promotes or inhibits the extracellular matrix degrading enzyme activity, and is obtainable by the method for screening according to the above (27) or the kit for screening according to the above (28),
(31) a diagnostic agent which comprises an antibody against a protein having an amino acid sequence identical or substantially identical to an amino acid sequence represented by SEQ ID No: 5, or a salt thereof,
(32) a method for detecting a proteoglycan degrading enzyme gene, which comprises mixing and culturing a transformant in which a test gene is introduced and an animal-derived cell producing cartilage or cartilage matrix, and measuring glycosaminoglycan sulfate in the culture supernatant,
(33) a method for screening for an agent for inhibiting or promoting the proteoglycan degrading enzyme activity, which comprises mixing and culturing (i) a recombinant in which a gene encoding a protein having the proteoglycan degrading enzyme activity is introduced, (ii) an animal-derived cell producing cartilage or cartilage matrix, and (iii) a test compound, and measuring glycosaminoglycan sulfate in the culture supernatant,
(34) a method for screening for an agent for inhibiting or promoting the proteoglycan degrading enzyme activity, which comprises mixing and culturing (i) an animal cell comprising (a) the DNA according to the above (7), or (b) a DNA comprising a DNA having a base sequence encoding a protein comprising an amino acid sequence identical or substantially identical to an amino acid sequence represented by SEQ ID No: 5, (ii) an animal-derived cell producing cartilage or cartilage matrix, and (iii) a test compound, and measuring an amount of glycosaminoglycon sulfate in the supernatant,
(35) a non-human mammal which has a DNA having a DNA having a base sequence encoding a protein having an amino acid sequence identical or substantially identical to an amino acid sequence represented by SEQ ID No: 2, or a its mutated DNA,
(36) the animal according to the above (35), which may express a protein having an amino acid sequence identical or substantially identical to an amino acid sequence represented by SEQ ID No: 2, and so forth.
Further, the present invention provides:
(37) the protein according to the above (1), wherein the amino acid sequence substantially identical to an amino acid sequence represented by SEQ ID No: 2 is an amino acid sequence having about 95% or more, preferably about 98% or more homology with an amino acid sequence represented by SEQ ID No: 2,
(38) the protein according to the above (1), wherein the amino acid sequence substantially identical to an amino acid sequence represented by SEQ ID No: 2 is (a) an amino acid sequence in which 1 to 5 (preferably, 1 to 3) amino acids in an amino acid sequence represented by SEQ ID No: 2 are deleted, (b) an amino acid sequence in which 1 to 5 (preferably, 1 to 3) amino acids are added to an amino acid sequence represented by SEQ ID No: 2, (c) an amino acid sequence in which 1 to 5 (preferably, 1 to 3) amino acids in an amino acid sequence represented by SEQ ID No: 2 are substituted with other amino acids, or (d) an amino acid sequence as a combination thereof,
(39) a DNA which comprises a DNA having a base sequence which hybridizes with a base sequence encoding the DNA according to the above (7) or (9) under the highly stringent conditions,
(40) a recombinant vector which comprises the DNA according to the above (39),
(41) a transformant transformed with the recombinant vector according to the above (40),
(42) a method for producing a protein encoded by the DNA according to the above (39) or a salt thereof, which comprises culturing the transformant according to the above (41), and producing and accumulating a protein encoded by the DNA according to the above (39), followed by collecting this,
(43) a protein encoded by the DNA according to the above (39) or a salt thereof, which is produced by the method for producing according to the above (42),
(44) the method for screening according to the above (19), which comprises measuring the protease activity (i) when a substrate is contacted with the protein according to the above (1), the partial peptide according to above (6) or a salt thereof, and the protease activity (ii) when a substrate and a test compound are contacted with the protein according to the above (1), the partial peptide according to the above (6) or a salt thereof, and comparing them,
(45) a medicine which comprises a compound or a salt thereof, which promotes the protease activity of the protein according to the above (1), the partial peptide according to the above (6) or a salt thereof, which is obtained by using the method for screening according to the above (19) or the kit for screening according to the above (20),
(46) the medicine according to the above (45), which is an agent for treating or preventing disc herniation, ischialgia, glomerular nephritis, diabetic nephropathy, hepatic fibrosis, pulmonary fibrosis or osteopetrosis,
(47) a medicine which comprises a compound or a salt thereof, which inhibits the protease activity of the protein according to the above (1), the partial peptide according to the above (6) or a salt thereof, which is obtained by the method for screening according to the above (19) or the kit for screening according to the above (20),
(48) the medicine according to the above (47), which is an agent for treating or preventing rheumatoid arthritis, osteoarthritis, osteoporosis, cancer, arteriosclerosis or corneae ulcer,
(49) a method for quantitating the protein according to the above (1), the partial peptide according to the above (6) or a salt thereof in a test solution, which comprises competitively reacting the antibody according to the above (14) with a test solution and the labeled protein according to the above (1), the partial peptide according to the above (4) or a salt thereof, and measuring a rate of the labeled protein according to the above (1), the partial peptide according to the above (4) or a salt thereof, which is bound to the antibody,
(50) a method for quantitating the protein according to the above (1), the partial peptide according to the above (6) or a salt thereof in a test solution, which comprises reacting simultaneously or successively a test solution with the antibody according to the above (14) insolubilized on a carrier and the labeled antibody according to the above (14) and, thereafter, measuring the activity of a label on the insolubilized carrier,
(51) a medicine which comprises the antibody according to the above (14),
(52) an antisense DNA which has a base sequence complementary or substantially complementary to the DNA according to the above (7), (9) or (39), and has the activity of being capable of inhibiting expression of the DNA,
(53) the antisense DNA according to the above (52), wherein the base sequence complementary or substantially complementary to the DNA according to the above (7), (9) or (39) is a base sequence having about 95% or more, preferably about 98% or more homology with a entire base sequence or a partial base sequence of a base sequence complementary to the DNA,
(54) a medicine which comprises the antisense DNA according to the above (52),
(55) a method for quantitating a protein or a salt thereof, comprising an amino acid sequence identical or substantially to an amino acid sequence represented by SEQ ID No: 5 in a test solution, which comprises competitively reacting an antibody against a protein comprising an amino acid sequence identical or substantially to an amino acid sequence represented by SEQ ID No: 5 or a salt thereof with a test solution and a labeled protein comprising an amino acid sequence identical or substantially to an amino acid sequence represented by SEQ ID No: 5 or a salt thereof, and measuring a ratio of the labeled protein containing amino acid identical or substantially to an amino acid sequence represented by SEQ ID No: 5 or a salt thereof which is bound to the antibody,
(56) a method for quantitating a protein comprising an amino acid sequence identical or substantially to an amino acid sequence represented by SEQ ID No: 5 or a salt thereof in a test solution, which comprises simultaneously or successively reacting a test solution with an antibody against a protein comprising an amino acid sequence identical or substantially to an amino acid sequence represented by SEQ ID No: 5 or a salt thereof which is insolubilized on a carrier and an antibody against a labeled protein comprising an amino acid sequence identical or substantially to an amino acid sequence represented by SEQ ID No: 5 or a salt thereof and, thereafter, measuring the activity of a label on the insolubilized carrier;
(57) a medicine which comprises an antibody against the protein comprising an amino acid sequence identical or substantially to an amino acid sequence represented by SEQ ID No: 5, or a salt thereof, and so forth.